Evaluation of candidate reference genes for expression study in Saccharum spp. hybrids under heavy metal stress

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1 Fujian Agriculture and Forestry University, Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fuzhou, Fujian, China
2 Fujian Agriculture and Forestry University, Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fu Zhou, Fu Jian, China
3 Key Lab of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China
DOI
10.7287/peerj.preprints.1487v1
Published
Accepted
Subject Areas
Molecular Biology, Plant Science
Keywords
heavy metal stress, Quantitative real-time PCR, Sugarcane, Reference gene
Copyright
© 2015 Ling et al.
Licence
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
Cite this article
Ling H, Huang N, Xu L, Huang L, Wu Q, Guo J, Su Y, Que Y. 2015. Evaluation of candidate reference genes for expression study in Saccharum spp. hybrids under heavy metal stress. PeerJ PrePrints 3:e1487v1

Abstract

Heavy metal contamination has been a significant problem limiting agricultural development, but sugarcane has recently emerged as a valuable phytoremediator. To better understand the molecular mechanism behind sugarcane's metal tolerance, it is necessary to analyze the expression of a novel gene(s) by qRT-PCR. Importantly, introducing internal reference gene(s) should be selected based upon gene stable expression, the inclusion of which could enhance both the accuracy and reliability of this method. In this study, 13 candidate genes were selected and evaluated stability of each genes. The results derived by statistical algorithms were then validated by normalizing the expression of metal related gene ScMTP (GenBank Accession No. KP864146), ScMT2-1-3 (GenBank Accession No. JQ627644), ScMPP (GenBank Accession No. CA267392.1) and ScHMA1 (GenBank Accession No. CA156665.1). Collectively, our qRT-PCR results indicated that in heavy metal-exposed sugarcane, APRT was the better single internal control in expression quantification. Moreover, the combination of CAC + CUL provide for a more accurate normalization for gene transcript profiles under these same conditions. The gene expression quantification that included APRT and CAC + CUL suggested that ScMTP had a differential expression pattern, ScMT2-1-3 and ScMPP were slightly inhibited, and ScHMA1 had minimal induction of expression in response to Cd2+ and Cu2+ stresses in sugarcane. Taken together, the suitable reference genes identified in this study will benefit future work aimed at the sugarcane gene functional characterization.

Author Comment

This is a submission to PeerJ for review.

Supplemental Information

Supplementary table S1 Stability evaluation of 13 candidate reference genes by four statistical algorithms in Saccharum spp

SV, stability value

DOI: 10.7287/peerj.preprints.1487v1/supp-1

Supplementary table S2 The general information of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 for quantitative real time PCR

DOI: 10.7287/peerj.preprints.1487v1/supp-2

Figure 1 The optimal combination of reference genes for gene expression normalization under heavy metal stresses in sugarcane

The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal combination of reference genes for accurate normalization in samples from different sugarcane cultivar samples.

DOI: 10.7287/peerj.preprints.1487v1/supp-3

Figure 2 Expression analysis of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 genes based on selected reference gene / genes under cadmium stress

ScMTP (Sugarcane metal tolerance protein gene, GenBank Accession No. KP864146), ScMT2-1-3 (Sugarcane metallothionein, GenBank Accession No. JQ627644), ScMPP (sugarcane metallophosphoesterase, GenBank Accession No. CA267392.1) and ScHMA1 (sugarcane heavy metal transporting ATPase 1, GenBank Accession No. CA156665.1) were heavy metal stress response genes in sugarcane. In this study, the normalization of ScMTP (A), ScMT2-1-3 (B), ScMPP (C) and ScHMA1 (D) employed a single reference gene, UBQ, APRT, GAPDH or 25S rRNA, or the reference gene set, CAC + CUL as reference control under cadmium chloride (CdCl2) treatment. Using 2-ΔΔCt to normalize the ScMTP, ScMT2-1-3, ScMPP and ScHMA1, the control sample were converted into 1. Significant different expression of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 were marked with p value (p < 0.01 level (highly significant) and p < 0.05 level (significant)) on the line respectively when comparing the normalization by UBQ, APRT, GAPDH and 25S rRNA with the normalization by CAC + CUL.

DOI: 10.7287/peerj.preprints.1487v1/supp-4

Figure 3 Expression analysis of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 genes based on selected reference gene / genes under copper stress

ScMTP (Sugarcane metal tolerance protein gene, GenBank Accession No. KP864146), ScMT2-1-3 (Sugarcane metallothionein, GenBank Accession No. JQ627644), ScMPP (sugarcane metallophosphoesterase, GenBank Accession No. CA267392.1) and ScHMA1 (sugarcane heavy metal transporting ATPase 1, GenBank Accession No. CA156665.1) were heavy metal stress response genes in sugarcane. In this study, the normalization of ScMTP (A), ScMT2-1-3 (B), ScMPP (C) and ScHMA1 (D) employed a single reference gene, UBQ, APRT, GAPDH or 25S rRNA, or the reference gene set, CAC + CUL as reference control under copper chloride (CuCl2) treatment. Using 2-ΔΔCt to normalize the ScMTP, ScMT2-1-3, ScMPPand ScHMA1, the control sample were converted into 1. Significant different expression of ScMTP, ScMT2-1-3, ScMPP and ScHMA1 were marked with p value (p < 0.01 level (2-tailed) and the 0.05 level (1-tailed) on the line respectively when comparing the normalization by UBQ, APRT, GAPDH and 25S rRNA wtih the normalization by CAC + CUL.

DOI: 10.7287/peerj.preprints.1487v1/supp-5